搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

The biological effects of low-dose ionizing radiation (LDIR) exposure in humans are not comprehensively understood,generating a high degree of controversy in published literature. The earliest stages of human development are known to be among the most sensitive to stress exposures,especially genotoxic stresses. However,the risks stemming from exposure to LDIR,particularly within the clinical diagnostic relevant dose range,have not been directly evaluated in human embryonic stem cells (hESCs). Here,we describe the dynamics of the whole genome transcriptional responses of different hESC lines to both LDIR and,as a reference,high-dose IR (HDIR). We found that even doses as low as 0.05 Gy could trigger statistically significant transient changes in a rather limited subset of genes in all hESCs lines examined. Gene expression signatures of hESCs exposed to IR appear to be highly dose-,time-,and cell line-dependent. We identified 50 genes constituting consensus gene expression signature as an early response to HDIR across all lines of hESC examined. We observed substantial differences in biological pathways affected by either LDIR or HDIR in hESCs,suggesting that the molecular mechanisms underpinning the responses of hESC may fundamentally differ depending on radiation doses. View Publication

Chromatin structure affects the extent of DNA damage and repair. Thus,it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here,we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC,heterochromatin is confined to distinct regions,while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives,normal human fibroblasts; and one cancer cell line. At the same time,the number of DNA repair foci were not statistically different among these cells. We showed that in hESC,DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells,and to a lesser extent in IMR90 normal fibroblasts,but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC. View Publication

Age-related macular degeneration (AMD) is one of the major causes of blindness in aging population that progresses with death of retinal pigment epithelium (RPE) and photoreceptor degeneration inducing impairment of central vision. Discovery of human induced pluripotent stem (hiPS) cells has opened new avenues for the treatment of degenerative diseases using patient-specific stem cells to generate tissues and cells for autologous cell-based therapy. Recently,RPE cells were generated from hiPS cells. However,there is no evidence that those hiPS-derived RPE possess specific RPE functions that fully distinguish them from other types of cells. Here,we show for the first time that RPE generated from hiPS cells under defined conditions exhibit ion transport,membrane potential,polarized vascular endothelial growth factor secretion,and gene expression profile similar to those of native RPE. The hiPS-RPE could therefore be a very good candidate for RPE replacement therapy in AMD. However,these cells show rapid telomere shortening,DNA chromosomal damage,and increased p21 expression that cause cell growth arrest. This rapid senescence might affect the survival of the transplanted cells in vivo and therefore,only the very early passages should be used for regeneration therapies. Future research needs to focus on the generation of safe" as well as viable hiPS-derived somatic cells." View Publication

Generating lentiviral vectors pseudotyped with different viral glycoproteins (GPs) may modulate the physicochemical properties of the vectors,their interaction with the host immune system,and their host range. We have investigated the capacity of a panel of GPs of both retroviral (amphotropic murine leukemia virus [MLV-A]; gibbon ape leukemia virus [GALV]; RD114,feline endogenous virus) and nonretroviral (fowl plague virus [FPV]; Ebola virus [EboV]; vesicular stomatitis virus [VSV]; lymphocytic choriomeningitis virus [LCMV]) origins to pseudotype lentiviral vectors derived from simian immunodeficiency virus (SIVmac251). SIV vectors were efficiently pseudotyped with the FPV hemagglutinin,VSV-G,LCMV,and MLV-A GPs. In contrast,the GALV and RD114 GPs conferred much lower infectivity to the vectors. Capitalizing on the conservation of some structural features in the transmembrane domains and cytoplasmic tails of the incorporation-competent MLV-A GP and in RD114 and GALV GPs,we generated chimeric GPs encoding the extracellular and transmembrane domains of GALV or RD114 GPs fused to the cytoplasmic tail (designated TR) of MLV-A GP. Importantly,SIV-derived vectors pseudotyped with these GALV/TR and RD114/TR GP chimeras had significantly higher titers than vectors coated with the parental GPs. Additionally,RD114/TR-pseudotyped vectors were efficiently concentrated and were resistant to inactivation induced by the complement of both human and macaque sera,indicating that modified RD114 GP-pseudotyped lentiviral vectors may be of particular interest for in vivo gene transfer applications. Furthermore,as compared to vectors pseudotyped with other retroviral GPs or with VSV-G,RD114/TR-pseudotyped vectors showed augmented transduction of human and macaque primary blood lymphocytes and CD34+ cells. View Publication

In order to develop a novel method of visualizing possible Ca(2+) signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters,we expressed the bioluminescent Ca(2+) reporter,apo-aequorin,in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca(2+) transients (generated by release from intracellular stores) were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2,indicating that holo-f-aequorin was functional in these cells. Furthermore,following the addition of exogenous ATP,an inositol trisphosphate receptor (IP3R) agonist,small Ca(2+) transients were generated from day 1 onward. That ATP was inducing Ca(2+) release from functional IP3Rs was demonstrated by treatment with 2-APB,a known IP3R antagonist. In contrast,following treatment with caffeine,a ryanodine receptor (RyR) agonist,a minimal Ca(2+) response was observed at day 8 of differentiation only. Thus,our data indicate that unlike RyRs,IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation. View Publication

Bispecific antibodies directed against tumor-associated target antigens and to surface receptors mediating T-cell activation,such as the TCR/CD3 complex and the costimulatory receptor CD28,are capable of mediating T-cell activation resulting in tumor cell killing. In this study,we used the B-cell-associated antigens CD19 and CD20 as target structures on human leukemic cells. We found that a combination of bispecific antibody fragments (bsFab2) with target x CD3 and target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in peripheral blood and bone marrow cultures from patients with chronic lymphocytic leukemia and follicular lymphoma. The bsFab2 targeting CD20 were considerably more effective than those binding to CD19. The colony-forming capacity of treated bone marrow was impaired due to large amounts of tumor necrosis factor alpha produced during bsFab2-induced T-cell activation. Neutralizing tumor necrosis factor alpha antibodies were found to reverse this negative effect without affecting T-cell activation and tumor cell killing. CD20 x CD28 bsFab2,when used alone rather than in combination,markedly improved the recognition of leukemic cells by allogeneic T cells. Therefore,these reagents may be capable of enhancing the immunogenicity of leukemic cells in general and,in particular,of increasing the antileukemic activity of allogeneic donor buffy coat cells in relapsed bone marrow transplanted patients. View Publication